The zona pellucida (ZP) is a thick extracellular coat that surrounds mammalian eggs. The mouse egg ZP consists of 3 glycoproteins, mZP1-3. One of the glycoproteins, mZP3, is recognized in a species-specific manner by free-swimming, acrosome-intact sperm when they bind to the ZP during fertilization. The sperm recognize and bind to specific Ser/Thr- linked (O-linked) oligosaccharides located at the mZP3 combining site for sperm. Binding to mZP3 induces sperm to undergo the acrosome reaction, a form of cellular exocytosis. Acrosome-reacted sperm then penetrate the ZP, reach the perivitelline space, and fuse with egg plasma membrane. In this application for support, experiments are described that address important questions about aspects of mZP3 structure and function. These aspects include the nature of essential residues at the mZP3 combining site for sperm and the molecular basis of species-specificity of mammalian sperm-egg interactions. A common experimental theme here is the use of embryonal carcinoma (EC) cells stably transfected with mutated mZP3 genes and of transgenic mice bearing an mZP3 null mutation. Null mutation mice were produced in our laboratory by targeted mutagenesis using homologous recombination in mouse embryonic stem (ES) cells. Eggs from homozygous (mZP3-/-) females lack a ZP and the animals are infertile. On the other hand, heterozygous females (mZP3+/-) and homozygous males (mZP3-/-) exhibit a wild-type phenotype and are fertile. Experiments described here use mutated mZP3 genes, evaluated in EC cells, and mZP3 null mutation mice to address fundamental aspects of mammalian fertilization. The studies proposed are a logical extension of previous work in our laboratory on mZP3. virtually all of the experimental techniques have been successfully in recent years in our laboratory. In view of the relatedness of mouse and human ZP3 it is anticipated that results of this investigation will impact significantly on our understanding of fundamental aspects of fertilization in human beings.